Mutations in the membrane-spanning domain of the human immunodeficiency virus envelope glycoprotein that affect fusion activity.

A chimeric protein consisting of the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) ectodomain joined to the transmembrane and cytoplasmic-tail domains of vesicular stomatitis virus G protein lost the ability to fuse CD4+ HeLa cells yet was transported to the cell surface and cleaved normally. These results suggested some critical role of the HIV gp41 transmembrane or cytoplasmic domain in fusion. Subsequent mutagenic analysis of the HIV-1 Env transmembrane domain revealed that the sequence of amino acid residues from positions 696 to 707 of the transmembrane domain was important for fusion function but was not required for anchoring of the Env protein in the lipid bilayer or for transport to the cell surface. Further analysis indicated that the basic residues at positions 696 and 707 were critical for membrane fusion activity, as was the spacing between these residues. These results demonstrate that in addition to providing an anchoring function, the specific amino acid sequence in the transmembrane domain plays a crucial role in the membrane fusion process.     

Functional analysis of the putative fusion domain of the baculovirus envelope fusion protein F

Group II nucleopolyhedroviruses (NPVs), e.g., Spodoptera exigua MNPV, lack a GP64-like protein that is present in group I NPVs but have an unrelated envelope fusion protein named F. In contrast to GP64, the F protein has to be activated by a posttranslational cleavage mechanism to become fusogenic. In several vertebrate viral fusion proteins, the cleavage activation generates a new N terminus which forms the so-called fusion peptide. This fusion peptide inserts in the cellular membrane, thereby facilitating apposition of the viral and cellular membrane upon sequential conformational changes of the fusion protein. A similar peptide has been identified in NPV F proteins at the N terminus of the large membrane-anchored subunit F(1). The role of individual amino acids in this putative fusion peptide on viral infectivity and propagation was studied by mutagenesis. Mutant F proteins with single amino acid changes as well as an F protein with a deleted putative fusion peptide were introduced in gp64-null Autographa californica MNPV budded viruses (BVs). None of the mutations analyzed had an major effect on the processing and incorporation of F proteins in the envelope of BVs. Only two mutants, one with a substitution for a hydrophobic residue (F152R) and one with a deleted putative fusion peptide, were completely unable to rescue the gp64-null mutant. Several nonconservative substitutions for other hydrophobic residues and the conserved lysine residue had only an effect on viral infectivity. In contrast to what was expected from vertebrate virus fusion peptides, alanine substitutions for glycines did not show any effect.     

Role of the specific amino acid sequence of the membrane-spanning domain of human immunodeficiency virus type 1 in membrane fusion

Fusion between cell and virus membranes mediated by gp41 initiates the life cycle of human immunodeficiency virus type 1. In contrast to the many studies that have elucidated the structure-function relationship of the ectodomain, the study of the membrane-spanning domain (MSD) has been rather limited. In particular, the role that the MSD's specific amino acid sequences may have in membrane fusion as well as other gp41 functions is not well understood. The MSD of gp41 contains well-conserved glycine residues that form the GXXXG motif (G, glycine; X, other amino acid residues), a motif often found at the helix-helix interface of membrane spanning alpha-helices. Here we examined the role that the specific amino acid sequence of the gp41 MSD has in gp41 function, particularly in membrane fusion, by making two types of MSD mutants: (i) glycine substitution mutants in which glycine residues of the MSD were mutated to alanine or leucine residues, and (ii) replacement mutants in which the entire MSD was replaced with one derived from glycophorin A or from vesicular stomatitis virus G. The substitution of glycines did not affect gp41 function. MSD-replacement mutants, however, showed severely impaired fusion activity. The assay using the Env expression vector revealed defects in membrane fusion after CD4 binding steps in the MSD-replacement mutants. In addition, the change in Env processing was noted for MSD-replacement mutants. These results suggest that the MSD of gp41 has a relatively wide but not unlimited tolerance for mutations and plays a critical role in membrane fusion as well as in other steps of Env biogenesis.     

An abundant evolutionarily conserved CSB-PiggyBac fusion protein expressed in Cockayne syndrome

Cockayne syndrome (CS) is a devastating progeria most often caused by mutations in the CSB gene encoding a SWI/SNF family chromatin remodeling protein. Although all CSB mutations that cause CS are recessive, the complete absence of CSB protein does not cause CS. In addition, most CSB mutations are located beyond exon 5 and are thought to generate only C-terminally truncated protein fragments. We now show that a domesticated PiggyBac-like transposon PGBD3, residing within intron 5 of the CSB gene, functions as an alternative 3′ terminal exon. The alternatively spliced mRNA encodes a novel chimeric protein in which CSB exons 1–5 are joined in frame to the PiggyBac transposase. The resulting CSB-transposase fusion protein is as abundant as CSB protein itself in a variety of human cell lines, and continues to be expressed by primary CS cells in which functional CSB is lost due to mutations beyond exon 5. The CSB-transposase fusion protein has been highly conserved for at least 43 Myr since the divergence of humans and marmoset, and appears to be subject to selective pressure. The human genome contains over 600 nonautonomous PGBD3-related MER85 elements that were dispersed when the PGBD3 transposase was last active at least 37 Mya. Many of these MER85 elements are associated with genes which are involved in neuronal development, and are known to be regulated by CSB. We speculate that the CSB-transposase fusion protein has been conserved for host antitransposon defense, or to modulate gene regulation by MER85 elements, but may cause CS in the absence of functional CSB protein.     

Role of the membrane-spanning domain of human immunodeficiency virus type 1 envelope glycoprotein in cell-cell fusion and virus infection

The membrane-spanning domain (MSD) of the human immunodeficiency virus type 1 (HIV-1) gp41 glycoprotein is critical for its biological activity. Previous C-terminal truncation studies have predicted an almost invariant core structure of 12 amino acid residues flanked by basic amino acids in the HIV-1 MSD that function to anchor the glycoprotein in the lipid bilayer. To further understand the role of specific amino acids within the MSD core, we initially replaced the core region with 12 leucine residues and then constructed recovery-of-function mutants in which specific amino acid residues (including a GGXXG motif) were reintroduced. We show here that conservation of the MSD core sequence is not required for normal expression, processing, intracellular transport, and incorporation into virions of the envelope glycoprotein (Env). However, the amino acid composition of the MSD core does influence the ability of Env to mediate cell-cell fusion and plays a critical role in the infectivity of HIV-1. Replacement of conserved amino acid residues with leucine blocked virus-to-cell fusion and subsequent viral entry into target cells. This restriction could not be released by C-terminal truncation of the gp41 glycoprotein. These studies imply that the highly conserved core residues of the HIV Env MSD, in addition to serving as a membrane anchor, play an important role in mediating membrane fusion during viral entry.     

Characterization of the prototype foamy virus envelope glycoprotein receptor-binding domain

The foamy virus (FV) glycoprotein precursor gp130(Env) undergoes a highly unusual biosynthesis, resulting in the generation of three particle-associated, mature subunits, leader peptide (LP), surface (SU), and transmembrane (TM). Little structural and functional information on the extracellular domains of FV Env is available. In this study, we characterized the prototype FV (PFV) Env receptor-binding domain (RBD) by flow cytometric analysis of recombinant PFV Env immunoadhesin binding to target cells. The extracellular domains of the C-terminal TM subunit as well as targeting of the recombinant immunoadhesins by the cognate LP to the secretory pathway were dispensable for target cell binding, suggesting that the PFV Env RBD is contained within the SU subunit. N- and C-terminal deletion analysis of the SU domain revealed a minimal continuous RBD spanning amino acids (aa) 225 to 555; however, internal deletions covering the region from aa 397 to 483, but not aa 262 to 300 or aa 342 to 396, were tolerated without significant influence on host cell binding. Analysis of individual cysteine point mutants in PFV SU revealed that only most of those located in the nonessential region from aa 397 to 483 retained residual binding activity. Interestingly, analysis of various N-glycosylation site mutants suggests an important role of carbohydrate chain attachment to N391, either for direct interaction with the receptor or for correct folding of the PFV Env RBD. Taken together, these results suggest that a bipartite sequence motif spanning aa 225 to 396 and aa 484 to 555 is essential for formation of the PFV Env RBD, with N-glycosylation site at position 391 playing a crucial role for host cell binding.     

Mutagenesis of the putative fusion domain of the Semliki Forest virus spike protein.

Semliki Forest virus (SFV), an alphavirus, infects cells via a low pH-triggered membrane fusion reaction that takes place within the cellular endocytic pathway. Fusion is mediated by the heterotrimeric virus spike protein, which undergoes conformational changes upon exposure to low pH. The SFV E1 spike subunit contains a hydrophobic domain of 23 amino acids that is highly conserved among alphaviruses. This region is also homologous to a domain of the rotavirus outer capsid protein VP4. Mutagenesis of an SFV spike protein cDNA was used to evaluate the role of the E1 domain in membrane fusion. Mutant spike proteins were expressed in COS cells and assayed for cell-cell fusion activity. Four mutant phenotypes were identified: (i) substitution of Gln for Lys-79 or Leu for Met-88 had no effect on spike protein fusion activity; (ii) substitution of Ala for Asp-75, Ala for Gly-83, or Ala for Gly-91 shifted the pH threshold of fusion to a more acidic range; (iii) mutation of Pro-86 to Asp, Gly-91 to Pro, or deletion of amino acids 83 to 92 resulted in retention of the E1 subunit within the endoplasmic reticulum; and (iv) substitution of Asp for Gly-91 completely blocked cell-cell fusion activity without affecting spike protein assembly or transport. These results argue that the conserved hydrophobic domain of SFV E1 is closely involved in membrane fusion and suggest that the homologous region in rotavirus VP4 may be involved in the entry pathway of this nonenveloped virus.     

Mutations within the putative membrane-spanning domain of the simian immunodeficiency virus transmembrane glycoprotein define the minimal requirements for fusion, incorporation, and infectivity

The membrane-spanning domain (MSD) of a number of retroviral transmembrane (TM) glycoproteins, including those from the human and simian immunodeficiency viruses (HIV and SIV), have been predicted to contain a charged arginine residue. The wild-type SIV TM glycoprotein is 354 amino acids long. The entire putative cytoplasmic domain of SIV (amino acids 193 to 354) is dispensable for virus replication in vitro, and such truncation-containing viruses are capable of reaching wild-type titers after a short delay. We show here that further truncation of eight additional amino acids to TM185 results in a protein that lacks fusogenicity but is, nevertheless, efficiently incorporated into budding virions. By analyzing a series of nonsense mutations between amino acids 193 and 185 in Env expression vectors and in the SIVmac239 proviral clone, a region of the SIV TM that contains the minimum requirement for glycoprotein-mediated cell-to-cell fusion and that for virus replication was identified. Virus entry and infectivity were evident in truncations to a minimum of 189 amino acids, whereas cell-cell fusion was observed for a protein of only 187 amino acids. Glycoprotein was efficiently incorporated into budding virions in truncations up to 185 amino acids, indicating that such proteins are membrane anchored and are transported to the cell surface. However, truncation of the TM to 180 amino acids resulted in a protein that displays a transport defect and may be retained in the endoplasmic reticulum. Based on our analyses of these mutants, an alternative model for the MSD of SIV is proposed. Our model suggests that membrane-imbedded charged residues can be neutralized by side-chain interactions with lipid polar head groups. As a consequence, the membrane-spanning region can be reduced by more than a helical turn. This new model accounts for the ability of truncations within the predicted MSD to remain membrane anchored and maintain biological activity.     

An evolutionarily conserved network of amino acids mediates gating in voltage-dependent potassium channels

A novel sequence-analysis technique for detecting correlated amino acid positions in intermediate-size protein families (50-100 sequences) was developed, and applied to study voltage-dependent gating of potassium channels. Most contemporary methods for detecting amino acid correlations within proteins use very large sets of data, typically comprising hundreds or thousands of evolutionarily related sequences, to overcome the relatively low signal-to-noise ratio in the analysis of co-variations between pairs of amino acid positions. Such methods are impractical for voltage-gated potassium (Kv) channels and for many other protein families that have not yet been sequenced to that extent. Here, we used a phylogenetic reconstruction of paralogous Kv channels to follow the evolutionary history of every pair of amino acid positions within this family, thus increasing detection accuracy of correlated amino acids relative to contemporary methods. In addition, we used a bootstrapping procedure to eliminate correlations that were statistically insignificant. These and other measures allowed us to increase the method's sensitivity, and opened the way to reliable identification of correlated positions even in intermediate-size protein families. Principal-component analysis applied to the set of correlated amino acid positions in Kv channels detected a network of inter-correlated residues, a large fraction of which were identified as gating-sensitive upon mutation. Mapping the network of correlated residues onto the 3D structure of the Kv channel from Aeropyrum pernix disclosed correlations between residues in the voltage-sensor paddle and the pore region, including regions that are involved in the gating transition. We discuss these findings with respect to the evolutionary constraints acting on the channel's various domains. The software is available on our website     

Incorporation of a charged amino acid into the membrane-spanning domain blocks cell surface transport but not membrane anchoring of a viral glycoprotein.

The membrane-spanning domain of the vesicular stomatitis virus glycoprotein (G protein) consists of a continuous stretch of 20 uncharged and mostly hydrophobic amino acids. We examined the effects of two mutations which change the amino acid sequence in this domain. These mutations were generated by oligonucleotide-directed mutagenesis of a cDNA clone encoding the G protein, and the altered G proteins were then expressed in animal cells. Replacement of an isoleucine residue in the center of this domain with a strongly polar but uncharged amino acid (glutamine) had no effect on membrane anchoring or transport of the protein to the cell surface. Replacement of this same isoleucine residue with a charged amino acid (arginine) generated a G protein that still spanned intracellular membranes but was not transported efficiently to the cell surface. The protein accumulated in the Golgi region in about 50% of the cells, and about 20% of the cells had detectable protein levels in a punctate pattern on the cell surface. In the remaining cells the protein accumulated in a vesicular pattern throughout the cytoplasm. Models which might explain the abnormal behavior of this protein are discussed.     

A single amino acid change in the Newcastle disease virus fusion protein alters the requirement for HN protein in fusion

The role of a leucine heptad repeat motif between amino acids 268 and 289 in the structure and function of the Newcastle disease virus (NDV) F protein was explored by introducing single point mutations into the F gene cDNA. The mutations affected either folding of the protein or the fusion activity of the protein. Two mutations, L275A and L282A, likely interfered with folding of the molecule since these proteins were not proteolytically cleaved, were minimally expressed at the cell surface, and formed aggregates. L268A mutant protein was cleaved and expressed at the cell surface although the protein migrated slightly slower than wild type on polyacrylamide gels, suggesting an alteration in conformation or processing. L268A protein was fusion inactive in the presence or absence of HN protein expression. Mutant L289A protein was expressed at the cell surface and proteolytically cleaved at better than wild-type levels. Most importantly, this protein mediated syncytium formation in the absence of HN protein expression although HN protein enhanced fusion activity. These results show that a single amino acid change in the F(1) portion of the NDV F protein can alter the stringent requirement for HN protein expression in syncytium formation.     

The membrane-spanning domain of gp41 plays a critical role in intracellular trafficking of the HIV envelope protein

Background

Each of the pathogenic human retroviruses (HIV-1/2 and HTLV-1) has a nonhuman primate counterpart, and the presence of these retroviruses in humans results from interspecies transmission. The passage of another simian retrovirus, simian foamy virus (SFV), from apes or monkeys to humans has been reported. Mandrillus sphinx, a monkey species living in central Africa, is naturally infected with SFV. We evaluated the natural history of the virus in a free-ranging colony of mandrills and investigated possible transmission of mandrill SFV to humans.

Results

We studied 84 semi-free-ranging captive mandrills at the Primate Centre of the Centre International de Recherches Médicales de Franceville (Gabon) and 15 wild mandrills caught in various areas of the country. The presence of SFV was also evaluated in 20 people who worked closely with mandrills and other nonhuman primates. SFV infection was determined by specific serological (Western blot) and molecular (nested PCR of the integrase region in the polymerase gene) assays. Seropositivity for SFV was found in 70/84 (83%) captive and 9/15 (60%) wild-caught mandrills and in 2/20 (10%) humans. The 425-bp SFV integrase fragment was detected in peripheral blood DNA from 53 captive and 8 wild-caught mandrills and in two personnel. Sequence and phylogenetic studies demonstrated the presence of two distinct strains of mandrill SFV, one clade including SFVs from mandrills living in the northern part of Gabon and the second consisting of SFV from animals living in the south. One man who had been bitten 10 years earlier by a mandrill and another bitten 22 years earlier by a macaque were found to be SFV infected, both at the Primate Centre. The second man had a sequence close to SFVmac sequences. Comparative sequence analysis of the virus from the first man and from the mandrill showed nearly identical sequences, indicating genetic stability of SFV over time.

Conclusion

Our results show a high prevalence of SFV infection in a semi-free-ranging colony of mandrills, with the presence of two different strains. We also showed transmission of SFV from a mandrill and a macaque to humans.     

Positive selection drives rapid evolution of certain amino acid residues in an evolutionarily highly conserved interferon-inducible antiviral protein of fishes

Viperin, an evolutionarily highly conserved interferon-inducible multifunctional protein, has previously been reported to exhibit antiviral activity against a wide range of DNA and RNA viruses. Utilizing the complete nucleotide coding sequence data of fish viperin antiviral genes, and employing the maximum likelihood-based codon substitution models, the present study reports the pervasive role of positive selection in the evolution of viperin antiviral protein in fishes. The overall rate of nonsynonymous (dN) to synonymous (dS) substitutions (dN/dS) for the three functional domains of viperin (N-terminal, central domain and C-terminal) were 1.1, 0.12, and 0.24, respectively. Codon-by-codon substitution analyses have revealed that while most of the positively selected sites were located at the N-terminal amphipathic α-helix domain, few amino acid residues at the C-terminal domain were under positive selection. However, none of the sites in the central domain were under positive selection. These results indicate that, although viperin is evolutionarily highly conserved, the three functional domains experienced differential selection pressures. Taken together with the results of previous studies, the present study suggests that the persistent antagonistic nature of surrounding infectious viral pathogens might be the likely cause for such adaptive evolutionary changes of certain amino acids in fish viperin antiviral protein.     

Identification of a membrane fusion domain and an oligomerization domain in the baculovirus GP64 envelope fusion protein.

The baculovirus GP64 envelope fusion protein (GP64 EFP) is the major envelope glycoprotein of the budded virion and has been shown to mediate acid-triggered membrane fusion both in virions and when expressed alone in transfected cells. Using site-directed mutagenesis and functional assays for oligomerization, transport, and membrane fusion, we localized two functional domains of GP64 EFP. To identify a fusion domain in the GP64 EFP of the Orgyia pseudotsugata multiple nuclear polyhedrosis virus (OpMNPV), we examined two hydrophobic regions in the GP64 EFP ectodomain. Hydrophobic region I (amino acids 223 to 228) is a cluster of 6 hydrophobic amino acids exhibiting the highest local hydrophobicity in the ectodomain. Hydrophobic region II (amino acids 330 to 338) lies within a conserved region of GP64 EFP that contains a heptad repeat of leucine residues and is predicted to form an amphipathic alpha-helix. In region I, nonconservative amino acid substitutions at Leu-226 and Leu-227 (at the center of the hydrophobic cluster) completely abolished fusion activity but did not prevent GP64 EFP oligomerization or surface localization. To confirm the role of region I in membrane fusion activity, we used a synthetic 21-amino-acid peptide to generate polyclonal antibodies against region I and demonstrated that antipeptide antibodies were capable of both neutralizing membrane fusion activity and reducing infectivity of the virus. In hydrophobic region II, mutations were designed to disrupt several structural characteristics: a heptad repeat of leucine, a predicted alpha-helix, or the local hydrophobicity along one face of the helix. Single alanine substitutions for heptad leucines did not prevent oligomerization, transport, or fusion activity. However, multiple alanine substitutions or proline (helix-destabilizing) substitutions disrupted both oligomerization and transport of GP64 EFP. In addition, a deletion that removed region II and the predicted alpha-helix was defective for oligomerization, whereas a larger deletion that retained region II and the predicted helix was oligomerized. These results indicate that region II is required for oligomerization and transport and suggest that the predicted helical structure of this region may be important for this function. Thus, by using mutagenesis, functional assays, and antibody inhibition, two functional domains were localized within the baculovirus GP64 EFP: a fusion domain located at amino acids 223 to 228 and an oligomerization domain located at amino acids 327 to 335 within a predicted amphipathic alpha-helix.     

Site-directed mutagenesis of conserved charged amino acid residues in ClpB from Escherichia coli

ClpB is a member of a multichaperone system in Escherichia coli (with DnaK, DnaJ, and GrpE) that reactivates strongly aggregated proteins. The sequence of ClpB contains two ATP-binding domains, each containing Walker consensus motifs. The N- and C-terminal sequence regions of ClpB do not contain known functional motifs. In this study, we performed site-directed mutagenesis of selected charged residues within the Walker A motifs (Lys212 and Lys611) and the C-terminal region of ClpB (Asp797, Arg815, Arg819, and Glu826). We found that the mutations K212T, K611T, D797A, R815A, R819A, and E826A did not significantly affect the secondary structure of ClpB. The mutation of the N-terminal ATP-binding site (K212T), but not of the C-terminal ATP-binding site (K611T), and two mutations within the C-terminal domain (R815A and R819A) inhibited the self-association of ClpB in the absence of nucleotides. The defects in self-association of these mutants were also observed in the presence of ATP and ADP. The four mutants K212T, K611T, R815A, and R819A showed an inhibition of chaperone activity, which correlated with their low ATPase activity in the presence of casein. Our results indicate that positively charged amino acids that are located along the intersubunit interface (this includes Lys212 in the Walker A motif of the N-terminal ATP-binding domain as well as Arg815 and Arg819 in the C-terminal domain) participate in intersubunit salt bridges and stabilize the ClpB oligomer. Interestingly, we have identified a conserved residue within the C-terminal domain (Arg819) which does not participate directly in nucleotide binding but is essential for the chaperone activity of ClpB.     

Truncation of the cytoplasmic domain induces exposure of conserved regions in the ectodomain of human immunodeficiency virus type 1 envelope protein

We have described a CD4-independent variant of HXBc2, termed 8x, that binds directly to CXCR4 and mediates CD4-independent virus infection. Determinants for CD4 independence map to residues in the V3 and V4-C4 domains together with a single nucleotide deletion in the transmembrane domain which introduces a frameshift (FS) at position 706. This FS results in a truncated cytoplasmic domain of 27 amino acids. We demonstrate here that while introduction of the 8x FS mutation into heterologous R5, X4, or R5X4 Env proteins did not impart CD4 independence, it did affect the conformation of the gp120 surface subunit, exposing highly conserved domains involved in both coreceptor and CD4 binding. In addition, antigenic changes in the gp41 ectodomain were also observed, consistent with the idea that the effects of cytoplasmic domain truncation must in some way be transmitted to the external gp120 subunit. Truncation of gp41 also resulted in the marked neutralization sensitivity of all Env proteins tested to human immunodeficiency virus-positive human sera and monoclonal antibodies directed against the CD4 or coreceptor-binding sites. These results demonstrate a structural interdependence between the cytoplasmic domain of gp41 and the ectodomain of the Env protein. They also may help explain why the length of the gp41 cytoplasmic domain is retained in vivo and may provide a way to genetically trigger the exposure of neutralization determinants in heterologous Env proteins that may prove useful for vaccine development.     

Amino acid residues within conserved domain VI of the vesicular stomatitis virus large polymerase protein essential for mRNA cap methyltransferase activity

During mRNA synthesis, the polymerase of vesicular stomatitis virus (VSV) copies the genomic RNA to produce five capped and polyadenylated mRNAs with the 5'-terminal structure 7mGpppA(m)pApCpApGpNpNpApUpCp. The 5' mRNA processing events are poorly understood but presumably require triphosphatase, guanylyltransferase, [guanine-N-7]- and [ribose-2'-O]-methyltransferase (MTase) activities. Consistent with a role in mRNA methylation, conserved domain VI of the 241-kDa large (L) polymerase protein shares sequence homology with a bacterial [ribose-2'-O]-MTase, FtsJ/RrmJ. In this report, we generated six L gene mutations to test this homology. Individual substitutions to the predicted MTase active-site residues K1651, D1762, K1795, and E1833 yielded viruses with pinpoint plaque morphologies and 10- to 1,000-fold replication defects in single-step growth assays. Consistent with these defects, viral RNA and protein synthesis was diminished. In contrast, alteration of residue G1674 predicted to bind the methyl donor S-adenosylmethionine did not significantly perturb viral growth and gene expression. Analysis of the mRNA cap structure revealed that alterations to the predicted active site residues decreased [guanine-N-7]- and [ribose-2'-O]-MTase activity below the limit of detection of our assay. In contrast, the alanine substitution at G1674 had no apparent consequence. These data show that the predicted MTase active-site residues K1651, D1762, K1795, and E1833 within domain VI of the VSV L protein are essential for mRNA cap methylation. A model of mRNA processing consistent with these data is presented.     

Study of the putative fusion regions of the preS domain of hepatitis B virus

In a previous study, it was shown that purified preS domains of hepatitis B virus (HBV) could interact with acidic phospholipid vesicles and induce aggregation, lipid mixing and leakage of internal contents which could be indicative of their involvement in the fusion of the viral and cellular membranes (Núñez, E. et al. 2009. Interaction of preS domains of hepatitis B virus with phospholipid vesicles. Biochim. Biophys. Acta 17884:417–424). In order to locate the region responsible for the fusogenic properties of preS, five mutant proteins have been obtained from the preS1 domain of HBV, in which 40 amino acids have been deleted from the sequence, with the starting point of each deletion moving 20 residues along the sequence. These proteins have been characterized by fluorescence and circular dichroism spectroscopy, establishing that, in all cases, they retain their mostly non-ordered conformation with a high percentage of β structure typical of the full-length protein. All the mutants can insert into the lipid matrix of dimyristoylphosphatidylglycerol vesicles. Moreover, we have studied the interaction of the proteins with acidic phospholipid vesicles and each one produces, to a greater or lesser extent, the effects of destabilizing vesicles observed with the full-length preS domain. The ability of all mutants, which cover the complete sequence of preS1, to destabilize the phospholipid bilayers points to a three-dimensional structure and/or distribution of amino acids rather than to a particular amino acid sequence as being responsible for the membrane fusion process.